Our long-standing goal has been to elucidate the molecular mechanisms of tRNA processing with the hope that this information would also reveal the biological significance of the complex events which intervene between transcription and ultimate function of RNA gene products. Using the yeast S. cerevisiae we have invoked an experimental strategy which has proven to be highly successful in its application to prokaryotic systems: the use of nonsense suppressors to determine the requirements for functional expression of a tRNA species by the isolation and characterization of mutants which have lost the suppressed phenotype. Yeast provides a unique opportunity to study tRNA processing in higher organisms; not only is it the only eukaryote with well documented tRNA suppressors, but it has been shown by us and others that a specific subset of tRNA genes are not colinear with the mature products. Our major attention has so far been directed at the identification of features of the precursor important for accurate and efficient splicing. We are particularly curious to know what role, if any, the intervening sequence (IVS) might play in its own removal. To do this, we have isolated mutants with reduced suppression by SUP4. By RNA sequence analysis of the accumulated precursor in one mutant, we have identified the mutation as an A yields G mutation at the 5' splice junction. Similar analyses of the mature tRNA showed that the IVS was nonetheless accurately excised. The 7-10x accumulation of the precursor argues that while the fidelity is maintained, the efficiency of splicing is significantly reduced. By comparison with other spliced precursors, it is suggested that this defect is consequent to an alteration in secondary (and/or tertiary) structure rather than nucleotide sequence per se.